Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

Acknowledgements AB conceived the project and wrote the manuscript. CVG conceived the experimental design. SW designed the GCaMP reporter. AM, KL, LV-M, SC and TB constructed strains and optimised imaging. MF developed the image analysis software. CVG and CP carried out the microfluidics experiments and imaging analysis. NG assisted with preparation of the manuscript. PS, SN and DMR developed and undertook the theoretical data analysis and contributed to the interpretation of the results. Funding AB, CG and TB were funded by the Wellcome Trust [Grant number 206412/A/17/Z]. AB and DR were supported by a Wellcome Trust Institutional Strategic Support Award (WT204909/Z/16/Z). CP was funded by a University of Exeter studentship (113516). This work was also supported by a Royal Society URF (UF080611), an MRC NIRG (G0900211/90671) and the MRC-Centre for Medical Mycology at the University of Exeter (MR/N006364/2). DR was funded by the Medical Research Council (MR/P022405/1). SN was supported by the Medical Research Council via the GW4 BioMed2 DTP (MR/W006308/1). MCA was supported by a European Commission ITN ‘FungiBrain’ studentship (607963). LL and SC were funded by a Wellcome Trust Institutional Strategic Support Award to the University of Aberdeen. NG acknowledges support of Wellcome Trust Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards (101873, 200208, 215599, 224323). NG and AB thank the MRC (MR/M026663/2) for support. This study/research is funded by the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.Peer reviewedPublisher PD

Murine model for Fusarium oxysporum invasive fusariosis reveals organ-specific structures for dissemination and long-term persistence

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Fungal iron availability during deep seated candidiasis is defined by a complex interplay involving systemic and local events

Peer reviewedPublisher PD

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordIntracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans, for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.Wellcome TrustUniversity of ExeterRoyal SocietyMedical Research Council (MRC)European CommissionNational Institute for Health and Care Research (NIHR

High-Throughput Screen for Identifying Small Molecules That Target Fungal Zinc Homeostasis

Resistance to traditional antifungal drugs has increased significantly over the past three decades, making identification of novel antifungal agents and new targets an emerging priority. Based on the extraordinary zinc requirement of several fungal pathogens and their well-established sensitivity to zinc deprivation, we developed an efficient cell-based screen to identify new antifungal drugs that target the zinc homeostasis machinery. The screen is based on the zinc-regulated transcription factor Zap1 of Saccharomyces cerevisiae, which regulates transcription of genes like the high-affinity zinc transporter ZRT1. We generated a genetically modified strain of S. cerevisae that reports intracellular zinc deficiency by placing the coding sequence of green fluorescent protein (GFP) under the control of the Zap1-regulated ZRT1 promoter. After showing that the GFP fluorescence signal correlates with low intracellular zinc concentrations in this strain, a protocol was developed for screening small-molecule libraries for compounds that induce Zap1-dependent GFP expression. Comparison of control compounds and known modulators of metal metabolism from the library reveals a robust screen (Z′ = 0.74) and validates this approach to the discovery of new classes of antifungal compounds that interfere with the intracellular zinc homeostasis. Given that growth of many pathogenic organisms is significantly impaired by zinc limitation; these results identify new types of antifungal drugs that target critical nutrient acquisition pathways

Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity

Background: The aim of this study was to investigate the possibility that a decreased mitochondrial ATP synthesis causes muscular and mental fatigue and plays a role in the pathophysiology of the chronic fatigue syndrome (CFS/ME).Methods: Female patients (n = 15) and controls (n = 15) performed a cardiopulmonary exercise test (CPET) by cycling at a continuously increased work rate till maximal exertion. The CPET was repeated 24 h later. Before the tests, blood was taken for the isolation of peripheral blood mononuclear cells (PBMC), which were processed in a special way to preserve their oxidative phosphorylation, which was tested later in the presence of ADP and phosphate in permeabilized cells with glutamate, malate and malonate plus or minus the complex I inhibitor rotenone, and succinate with rotenone plus or minus the complex II inhibitor malonate in order to measure the ATP production via Complex I and II, respectively. Plasma CK was determined as a surrogate measure of a decreased oxidative phosphorylation in muscle, since the previous finding that in a group of patients with external ophthalmoplegia the oxygen consumption by isolated muscle mitochondria correlated negatively with plasma creatine kinase, 24 h after exercise.Results: At both exercise tests the patients reached the anaerobic threshold and the maximal exercise at a much lower oxygen consumption than the controls and this worsened in the second test. This implies an increase of lactate, the product of anaerobic glycolysis, and a decrease of the mitochondrial ATP production in the patients. In the past this was also found in patients with defects in the mitochondrial oxidative phosphorylation. However the oxidative phosphorylation in PBMC was similar in CFS/ME patients and controls. The plasma creatine kinase levels before and 24 h after exercise were low in patients and controls, suggesting normality of the muscular mitochondrial oxidative phosphorylation.Conclusion: The decrease in mitochondrial ATP synthesis in the CFS/ME patients is not caused by a defect in the enzyme complexes catalyzing oxidative phosphorylation, but in another factor

Candida albicans Infection of Caenorhabditis elegans Induces Antifungal Immune Defenses

Candida albicans yeast cells are found in the intestine of most humans, yet this opportunist can invade host tissues and cause life-threatening infections in susceptible individuals. To better understand the host factors that underlie susceptibility to candidiasis, we developed a new model to study antifungal innate immunity. We demonstrate that the yeast form of C. albicans establishes an intestinal infection in Caenorhabditis elegans, whereas heat-killed yeast are avirulent. Genome-wide, transcription-profiling analysis of C. elegans infected with C. albicans yeast showed that exposure to C. albicans stimulated a rapid host response involving 313 genes (124 upregulated and 189 downregulated, ∼1.6% of the genome) many of which encode antimicrobial, secreted or detoxification proteins. Interestingly, the host genes affected by C. albicans exposure overlapped only to a small extent with the distinct transcriptional responses to the pathogenic bacteria Pseudomonas aeruginosa or Staphylococcus aureus, indicating that there is a high degree of immune specificity toward different bacterial species and C. albicans. Furthermore, genes induced by P. aeruginosa and S. aureus were strongly over-represented among the genes downregulated during C. albicans infection, suggesting that in response to fungal pathogens, nematodes selectively repress the transcription of antibacterial immune effectors. A similar phenomenon is well known in the plant immune response, but has not been described previously in metazoans. Finally, 56% of the genes induced by live C. albicans were also upregulated by heat-killed yeast. These data suggest that a large part of the transcriptional response to C. albicans is mediated through “pattern recognition,” an ancient immune surveillance mechanism able to detect conserved microbial molecules (so-called pathogen-associated molecular patterns or PAMPs). This study provides new information on the evolution and regulation of the innate immune response to divergent pathogens and demonstrates that nematodes selectively mount specific antifungal defenses at the expense of antibacterial responses

Додатковий том «Словника української мови»

У статті подано історію роботи над Додатковим томом «Словника української мови» в 11-ти томах, описано джерела наповнення реєстру, структуру словникових статей, наведено приклади розробки статей різного типу – як нововведених слів, так і таких, що були в «Словнику української мови» і зазнали доповнення. Завдання лексикографів, які працювали над Додатковим томом, – відобразити динаміку лексичного шару української мови 1980-их рр. ХХ ст. – початку ХХІ ст. з акцентуванням її інноваційних й актуалізованих аспектів

Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.

The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity